ddPCR OPTIMIZATION AND DEVELOPMENT OF MULTIPLEX MODULES FOR ANALYZING EVENTS OF GENETIC MODIFICATION IN MAIZE

Abstract

The planting, marketing and consumption of transgenic maize is a matter of debate in Mexico, since it is a centre of origin and diversification of native maize varieties and breeds. The droplet digital polymerase chain reaction (ddPCR) has proven to be more efficient than the gold standard test (qPCR) for the analysis of genetically modified maize. Therefore, the objective was to optimize ddPCR tests for transgene analysis in maize and to generate multiplex detection modules. The applicability of the primers was evaluated by bioinformatics analysis. Then, using gradients, an optimal hybridization and elongation temperature was determined and the primers concentration, droplet recovery and the “rain” effect were evaluated. Optimization was verified by duplex testing, and finally multiplex modules were developed. The temperature that allowed a clear separation between positive and negative droplet clusters for the 11 simplex tests was 58.5 °C, with relatively low concentrations of primers and probes. The recovered droplets were adequate for the purpose of the analyses and the “rain” obtained was irrelevant. In the duplex tests, the potential for quantification was confirmed without using calibration curves; most assays agreed with validation reports or with theoretical percentages of genetic variability. Finally, in the multiplex modules, variations in primers and probe concentration were performed to differentiate clusters of positive droplets in a single detector channel. The best differences were achieved at concentrations with 1:3 differences. The results showed that the optimized tests are suitable for the detection and possible quantification of 10 transgenes in maize.